Highly Selective Drug-Derived Fluorescent Probes for the Cannabinoid Receptor Type 1 (CB1R)

The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R’s cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.


■ INTRODUCTION
Present in all vertebrates, the cannabinoid receptor type 1 (CB 1 R), alongside the cannabinoid receptor type 2 (CB 2 R), is the key signal transducer of the endocannabinoid system (ECS). 1 CB 1 R is predominantly expressed on presynaptic terminals in the central nervous system (CNS), where it modulates neuronal signaling. 2,3Yet, CB 1 R was also found on peripheral cells and organs. 4,5In conjunction with its localization, CB 1 R has implications in the homeostasis of various fundamental physiological processes, such as appetite regulation, 6 energy metabolism, 7 synaptic plasticity, 8 and nociception. 9Most relevant is the fact that the aberrant expression of CB 1 R is associated with pathophysiological processes, among which are neurodegenerative diseases and neurological, metabolic, and inflammatory disorders. 10,11This plethora of potential therapeutic indications underlines the clinical relevance and has triggered extensive pharmaceutical research on CB 1 R. 12,13 However, the withdrawal of the inverse agonist Rimonabant (6, Figure 2) as an anti-obesity agent from the European market in 2008 represented a major incision in CB 1 R drug research. 14,15The complexity of ECS signaling and the CB 1 R-related CNS side effects have called for appropriate analytical tools to advance a deeper understanding of the involvement of the CB 1 R in the ECS. 16For translation of novel promising CB 1 R drug candidates 17−24 emerging from preclinical studies to clinical trials, visualization tools for spatiotemporally resolved CB 1 R pharmacological characterization are urgently required. 25luorescence-based techniques have evolved into a powerful method for studying G-protein coupled receptors (GPCRs). 26,27In particular, small molecule fluorescent probes represent versatile tools to elucidate various mechanistic aspects of GPCR pharmacology.−33 While several CB 2 R fluorescent probes were recently reported, only a few CB 1 R fluorescent probes have been described so far. 34,35The only two examples of CB 1 R imaging probes are phytocannabinoid-derived (1 and 2, Figure 1). 36,37In general, issues associated with phytocannabinoid probes are their limited selectivity over CB 2 R and lipophilicity that may result in a high unspecific background signal.Besides phytocannabinoids, synthetic drug-derived fluorescent probes have been reported (e.g., 3, Figure 1).However, their selective CB 1 R imaging application was not validated further. 30,38,39In turn, no CB 1 Rselective imaging probe is described that has been unambiguously characterized pharmacologically in terms of its functional activity and selectivity profile.However, knowledge of the detailed mechanism of action of an imaging probe is crucial to obtain definite and relevant biological results on live cells, as the probe represents a pharmacologically active unit itself.For example, GPCR agonists may induce receptor internalization relevant for internalization studies, whereas an inverse agonist may allow for the detection of steady-state membrane receptor pools. 40erein, we report the modular design, synthesis, pharmacological evaluation, and application of CB 1 R-selective fluorescent probes.The probes were conceptualized based on a reverse design approach employing synthetic drug-like CB 1 R ligands with a defined pharmacological profile as starting points. 41By this way, we capitalized for the construction of high-quality tailored labeled probes from drug-derived validated starting points and the analysis and consideration of pre-existent structure-activity relationship (SAR).This study led to the discovery of novel and highly selective pyrrole-based CB 1 R fluorescent probes.Further exploration showcased the versatility of these inverse agonist fluorescent probes for pharmacological time-resolved Forster resonance transfer (TR-FRET) studies and CB 1 R imaging on live cells.Our approach presents a viable design concept for future CBR probes leveraging a deeper understanding of CB 1 R pharmacology.

■ DESIGN CONCEPT
−45 As an amino acid, the DEG motif has granted a high flexibility and synthetic simplicity for amide bond-based derivatization by different pharmacophoric units and linkers achieving CB 2 R probes.Considering the high homology of CB 1 R and CB 2 R, we concluded that this DEG motif would be also a suitable centerpiece in conjunction with a CB 1 R fluorescent probe.Analysis of CB 1 R ligand SAR and ligand alignment studies indicated a strong preference for similar branched lipophilic α,α-diethyl substitutions, providing the necessary steric bulk and favoring distinct conformational preorientation. 46herefore, we aimed to expand the design scope of this privileged and chemically stable DEG-based probe design toward a CB 1 R probe platform (4, Figure 2A).Attempting this, candidate structures as pharmacophore donors were selected among six high-affinity drug-like CB 1 R ligands (5− 10, Figure 2B).−52 The probe design was based on three exploration steps to achieve validation of our construction concept.We first replaced the original apolar amine unit in 5−10 with DEG ethyl ester to examine whether this modification would be tolerated (Figure 2C).Ideally, the pharmacological properties of the original CB 1 R ligands, such as high affinity, functional activity, and selectivity for CB 1 R, would be preserved upon these structural changes.In the second and third step, the influence of linker attachment and then of fluorescent dye installation was investigated, respectively (Figure 2C).The SAR was screened throughout the series with pharmacological characterization of binding affinity to CB 1 R and CB 2 R.

■ RESULTS AND DISCUSSION
Chemistry.The synthesis of the novel DEG ethyl ester ligands 14−19 is outlined in Scheme 1A.The synthesis began with SOCl 2 -facilitated esterification of the carboxylic acid functional group of 11, followed by protection of the amino  30,36,37,39 Journal of Medicinal Chemistry Figure 2. α,α-Diethyl glycine (DEG) amide probe design approach.(A) General construction scheme for CB 1 R fluorescent probes based on DEG.(B) Selected drug-like CB 1 R ligands 47−52 bearing amide bonds are useful as donors for CB 1 R pharmacophoric units (blue) for the attachment to the DEG centerpiece.Amine fragments (black) were replaced with DEG.(C) Exemplified three-step probe exploration for CB 1 R pyrrole-based fluorescent probes 28 and 29.
group to give benzylidene intermediate 12.The central DEG building block 13 was obtained via an alkylation of 12 using ethyl iodide and KHDMS, followed by hydrolysis of the benzylidene imine under acidic conditions.HATU-mediated amide coupling reaction with respective carboxylic acids 42−47 furnished the desired DEG ligands 14−19.
To determine the optimal linker length for the fluorescent dye attachment, commercially available N-Fmoc-α,α-diethyl glycine 20 was utilized (Scheme 1B).Using an orthogonal protecting For synthesis of probes 51 and 52, see the Experimental Section. 54roup strategy, a series of N-Boc protected diamine linkers (n = 1−4) were coupled to amino acid 20 using HATU to give access to 21a−d.Fmoc-protecting group removal of compounds 21a− d using DBU was followed by in situ coupling to corresponding carboxylic acids 42−47 to afford Boc-protected congeners 22a− c, 23a−d, and 24−27.Notably, the HATU coupling of 42 with Fmoc-deprotected 22a−c resulted in consistently low yields with an unreactive HOAt-ester intermediate as the main product (S53, see Figure S24).This observation could be attributed to the steric hindrance of DEG, which is known to be a challenging factor in amide couplings. 53The initially observed low yields of <10% for the amide coupling reaction (see 22a) were improved for 22b and 22c by increasing the temperature to 40−45 °C and prolongation of the reaction times to 4−7 days (56 and 48% yield, respectively).
To obtain target fluorescent probes 28−37 and 40−41, the terminal N-Boc protecting group of -22b, 23b, and 24−27 had to be removed (Scheme 1C).Cleavage using TFA was applied for 23b, 24, 26, and 27.This procedure, however, was not compatible with compounds 22b and 25 where partial degradation in the presence of TFA was observed.To overcome this problem, Boc-deprotection of 22b was performed under mild, microwave-assisted cleavage using 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). 55This procedure was found to be mild enough to avoid decomposition and yielded the free terminal amine of 22b.The resulting free amines were coupled either to carboxy 5/6-tetramethyl rhodamine (TAMRA) fluorescent dye by amide coupling or to fluoro-nitrobenzoxadiazole (F-NBD) via nucleophilic aromatic substitution conditions to achieve probes 28−37 (see Figures S25−S34).Boc-deprotection of 25 was possible neither with TFA nor under HFIP/MW conditions.Therefore, probes 40 and 41 were synthesized via a variation of the synthetic route starting with Boc-deprotection of 21b followed by conjugation of fluorescent dyes to obtain intermediates 38 and 39.After the removal of the Fmocprotecting group with DBU, another amide coupling under HATU conditions gave access to the fluorescent probes 40 and 41 (Scheme 1C, see Figures S35 and S36).
Computational Studies.Docking studies were conducted to explore the orientation of novel DEG ethyl ester ligands 14− 19.Exemplified in Figure 3A is the docking structure of the DEG ethyl ester 14 derived from 5 in inactive CB 1 R (utilizing PDB ID: 5TGZ). 56Interestingly, the pharmacophoric pyrrole unit in 14 bearing the DEG centerpiece unit was well accommodated in the binding pocket of CB 1 R aligning with the known cocrystallized ligand AM6538 (PDB ID: 5TGZ) (see Figure S14).The DEG unit in 14 was oriented toward the extracellular space comparable with the piperidine unit of AM6538.Docking poses of compounds 15−19 consistently showed that the ethyl ester moiety points toward the N-terminus of CB 1 R (see Supporting Information, Figures S9−S13) and that the α-ethyl side chains are favorably involved in attractive van der Waals interactions with the F174 side chain.We therefore concluded that DEG can favorably replace the original amine units of 5−10 (Figure 2B, black fragments).Hence, utilization of the CB 1 R pharmacophoric units from 5−10 in conjunction with a DEG centerpiece appeared as a promising approach toward a platform for CB 1 R fluorescent probes.In addition, the docking study revealed that the terminal carboxy group of DEG is an ideal linker attachment site, allowing free access to the extracellular space, thereby avoiding extensive linker attachment studies (Figure 3A,B).
To estimate a proper linker length for fluorescent dye attachment in our probes, docking studies were performed on compounds 22b, 23b, and 24−27 in the same receptor structure.The docking pose of 22b is shown in Figure 3C (for compounds 23b and 24−27, see Figures S9−S13).The PEG chain with n = 2 was predicted to reach out to the CB 1 R extracellular site through the trans-membrane helices TM1 and 2. This linker appeared to be long enough to allow for the envisioned fluorescent dye attachment at the terminal amine without interfering with binding (Figure 3D).A detailed SAR investigation on the linker length confirmed these results (see the next section).
In Vitro Pharmacology.Pharmacological Profiling of DEG Ethyl Ester Intermediates.We first analyzed the novel drug-like DEG ester-derived CB 1 R ligands 14−19 to experimentally examine whether the insertion of DEG moiety would be tolerated without compromising CB 1 R affinity and functional activity compared to the original counterparts 5−10 (Table 1, binding data with standard error of mean in Table S1).The binding affinities were measured in a radioligand binding assay on Chinese hamster ovary (CHO) membranes stably expressing either human CB 1 R or CB 2 R. In this assay, all compounds (14− 19) exhibited nanomolar to submicromolar affinity for human CB 1 R.However, among all tested chemotypes, only 14 preserved CB 1 R affinity and showed marked selectivity for Notably, compounds 17 and 18 showed a swap from CB 1 R-selectivity to CB 2 R-selectivity.This finding could be attributed to the acquired structural similarity to 3,4,5-substituted pyridine CB 2 R-ligands 43 upon conjugation with the DEG ethyl ester.Even though the differences between the CB 1 R and CB 2 R binding affinities for compounds 15 and 16 were not pronounced, they exhibited a slight preference for CB 2 R.Even though indazole-based 19 showed no CB 1 Rselectivity after installation of the DEG moiety, the lack of CB 1 Rselectivity was not surprising in this case as 19 was derived from  agonist 10, which already featured a weak CB 1 R preference [K i (CB 2 R)/K i (CB 1 R) = 4-fold selectivity] commonly observed with this compound class. 52,57In a CB 1 R cAMP functional homogeneous time-resolved fluorescence (HTRF) assay, 58 14, 15, 17, and 18 were found to be inverse agonists and 19 an agonist, while 16 showed no activity in the assay.To our delight, all DEG esters retained the functional activity of their original structures 5−10.
−61 Hence, to get an unbiased and detailed picture of the linker tolerance of the structures and optimal length for fluorescent dye conjugation, we progressed with a linker screen by using compound series 22a−c and 23a−d with N-Boc-protected terminus as model compounds.
The pharmacological evaluation of the DEG probe precursors 22a−c, 23a−d, and 24−27 is outlined in Table 2 (binding data with standard error of mean in Table S1).Even though the overall binding affinities of compounds 22a−c declined compared to 14, CB 1 R preference was preserved.Despite the absence of a linear correlation between the linker length and binding affinities, linker length n = 2 of 22b appeared as most favorable, as it exhibited the highest CB 1 R affinity and selectivity.In addition, 22b retained inverse agonist activity (IC 50  = 131  nM, E max = −69%).This linker selection was supported by our docking studies (Figure 3).We further examined the effect of the linker attachment and length on pyrazoles 23a−d compared to DEG ethyl ester ligand 15.Interestingly, while attachment of DEG ester in compound 15 attenuated its CB 1 R selectivity, installation of N-Boc-protected PEG chains in compounds 23a− d revived the CB 1 R-selectivity over CB 2 R. Unlike 22a−c, compounds 23a−d showed a linear correlation between CB 1 R affinity and the linker lengths.In this series, 23a (n = 1) showed the highest affinity and selectivity to CB 1 R.However, a short linker might lead to a steric clash with the receptor's binding pocket after the envisioned fluorescent dye installation and consequently compromise binding affinity.Altogether, the molecular docking of 22b and binding data of series 22a−c and 23a−d supported the selection of n = 2 as the most suited linker for our probes.To our delight, 23b also showed conserved  H]CP55,940 displacement assays on CHO cell membranes stably expressing human CB 1 R or human CB 2 R. Values are means of three independent experiments performed in duplicate.b The activity levels (IC 50 ) of 22b and 23b were measured using cells stably expressing hCB 1 R in homogeneous time-resolved fluorescence (HTRF) cAMP assay.The data are the means of three independent experiments performed in technical replicates.c Maximum effect (E max in %) was normalized to reference full agonist CP55,940.n.d. is not determined.All data with standard error of mean are given in the Supporting Information.functional activity as an inverse agonist on CB 1 R (IC 50  = 64.6  nM, E max = −44%).
The N-Boc-protected PEG chain with n = 2, as the ideal linker, was also examined in combination with pharmacophores 44−47 yielding DEG probe precursors 24−27.Unfortunately, compounds 24−27 exhibited no or significantly weaker CB 1 R binding (between 3 and >10 μM) (Table 2) and instead CB 2 R preference indicating that linker elongation is not equally well tolerated by all pharmacophores.
Pharmacological Profiling of CB 1 R Fluorescent Probes.Next, we studied the CB 1 R binding affinity of probes 28−37 and 40−41 equipped with fluorescent dyes NBD and TAMRA.As the presence of a fluorescent dye might significantly alter the pharmacological profile of the probes, 59,62,63 we thoroughly characterized our target compounds (Table 3, binding data with Table 3. Binding Affinities and Functional Activity of the Fluorescent Probes a K i (nM) values obtained from [ 3 H]CP55,940 displacement assays on CHO membranes stably expressing human CB 1 R or human CB 2 R. Values are means of three independent experiments performed in duplicate.b The activity levels (IC 50 ) of 28−30 were measured using cells stably expressing hCB 1 R in homogeneous time-resolved fluorescence (HTRF) cAMP assay.The data are the means of three independent experiments performed in technical replicates.c Maximum effect (E max in %) was normalized to reference full agonist CP55,940.n.d. is not determined.All data with standard error of mean are given in the Supporting Information.
standard error of mean in Table S1).In this study, we have chosen green-emitting NBD and orange-emitting TAMRA as examples for sterically small and large fluorescent dye, respectively.In addition, TAMRA as a partially zwitterionic hydrophilic rhodamine-derivative should be especially suited for cellular imaging of membrane proteins due to its good photostability and quantum yield.Photophysical characteristics of the probes were assessed in PBS buffer (Table S5).We determined the CB 1 R and CB 2 R binding profile of the labeled probes carrying different fluorescent dyes in the radioligand binding assay and in the functional HTRF cAMP assay.We observed fluorescent dye-dependent differences in the binding profile of the probes.For example, pyrrole-based probes 28 and 29 bearing NBD and TAMRA, respectively, maintained their CB 1 R-selectivity.However, the substantially lower K i value for the TAMRA probe [29, 8] suggested that the larger TAMRA dye might interfere with ligand binding, while NBD conjugation turned out to be beneficial for the CB 1 R affinity [28, K i (hCB 1 R) = 97 nM, K i (CB 2 R)/K i (CB 1 R) > 103] when compared to the DEG probe precursor 22b (K i (hCB 1 R) = 811 nM).In contrast to the binding assay, both inverse agonists 28 (IC 50 = 16.6 nM) and 29 (IC 50 = 102 nM) were more potent in the cAMP functional assay when compared to 22b and with only weak fluorescent dyedependency.A similar effect was observed for pyrazole-based probes 30 and 31.Inverse agonist NBD probe 30 50  = 60.3 nM] preserved its CB 1 R profile when compared to precursor 23b while TAMRA conjugation was deleterious for the binding affinity of 40 to either of the CBRs.To our surprise, the indazole-based NBD probe 36 showed binding to CB 1 R [K i (CB 1 R) = 1174 nM], while its DEG probe precursor 27 and TAMRA congener 37 were solely CB 2 R binders.Yet, both showed preferred binding to CB 2 R. Similarly, rigidified pyrazole 32, pyridine 40, and pyrazine 34 NBD probes displayed CB 2 R selectivity.Within this series, fluorescent dye dependency was observed again, as with their respective TAMRA congeners, 33, 41, and 35 did not bind to either of the receptors.
In summary, all NBD probes maintained CBR preference as observed with their corresponding DEG probe precursor structures in the linker screen (Table 2).In turn, installation of the sterically more demanding TAMRA dye was not tolerated well and led to a loss of CB 1 R binding affinity except for probe 29.This trend was further confirmed with two other small fluorescent dyes of the "Scotfluor" series 54 (CB 1 R-selective probes 51 and 52, for synthesis, see Experimental Section).Our investigation exemplifies that the modular reverse design approach is capable of facilitating and guiding the construction of DEG-based fluorescent probes from CB 1 R pharmacophores but that careful pharmacological characterization is crucial for probe design.
Conformational Molecular Dynamics Simulation.While the classical construction principle of fluorescent dye labeled probes features several physicochemical characteristics that might hamper passive cellular permeation, we still observed rather efficient permeation at low concentration of probe 29 in the confocal imaging experiment (vide infra).−67 We therefore investigated the unexpected membrane permeability of 29 by molecular dynamics (MD) simulations.
−70 During a 50 ns MD simulation, we analyzed the conformations of 29, their 3D PSA, and the amount of formed IMHBs in water and n-octane (as a model of apolar cell membrane environment).
Probe 29 adopted a broad range of conformations with variable 3D PSA (see Figures 4B and S15−S22).Consistently, the transition of compound 29 from water to n-octane would lead to a significant reduction of the mean 3D PSA and an increased number of IMHBs interactions, with the only exception being the spirolactone 6-isomer.For instance, the mean 3D PSA of the 5-zwitterion isomer (prevalent in water) would be reduced from 171.7 to 99.8 Å 2 when transitioning into n-octane and equilibrating into the spirolactone form (prevalent in apolar solvents).Simultaneously, the mean number IMHB of 0.1 in water would increase to 2.2 in n-octane (for other values see Table S4).
These MD data suggest that in particular, the 5-isomer of 29 has a strong tendency for chameleonic effects.In addition, based on the 3D PSA, a better membrane permeability by passive diffusion of probe 29 can be concluded than predicted by classical metrics of drug-likeness. 71This shows that MD-derived studies for assessment of intracellular accessibility of high molecular weight compounds are relevant and useful also for fluorescent probe conjugates. 68,72R-FRET Binding Assay.TR-FRET has evolved as an attractive alternative to radioligand binding assays using fluorescent probes as tracers.TR-FRET assays are available for CB 1 R 73−75 and especially suited for the determination of kinetic ligand−receptor interactions. 76,77Consequently, human embryonic kidney (HEK293TR) cells overexpressing SNAP-tagged hCB 1 R were labeled with a SNAP-Lumi4-Tb FRET-donor and cell membrane preparations generated.Laser excitation of the terbium cryptate (337 nm) on the N-terminus of CB 1 R induces energy transfer to a fluorescent probe when bound to CB 1 R.
We first examined saturation and kinetic binding parameters of TAMRA probe 29 on CB 1 R membrane preparations.The probe showed stable binding to the receptor over a time course of 30 min (Figure 5A).The saturation binding affinity value of 29 was lower (K D = 335.5 nM) (Figure 5B) than obtained in the radioligand binding assay, yet, still in a commensurate range.In a kinetic association and dissociation experiment, 29 exhibited a moderate association rate of 0.81 × 10 6 M −1 min −1 on hCB 1 R which supports its applicability as imaging probe (Table 5).
Exploring the binding kinetics of a ligand is a crucial aspect of GPCR drug development and can be used to promote improved drug efficacy. 78Using 29 as a fluorescent tracer, the kinetic parameters k on and k off and resulting K D of 6 and HU210 were determined (Table 5).In addition, their equilibrium binding affinity was determined with both fluorescent NBD 28 and TAMRA 29 as tracers in a simple competition binding assay (Figure 5C,D and Table 5).Competition binding affinities K i of the known CB 1 R ligands were in excellent agreement with the kinetic K D and literature radioligand binding affinities determined at human CB 1 R. 79−82 In addition, the determined K i values of 6 and HU210 were probe-independent.These experiments underscore the applicability of our fluorescent pyrazole probes 28 and 29 as highly useful tools in TR-FRETbased CB 1 R drug discovery to characterize the kinetic binding and equilibrium affinities of CB 1 R ligands in a potential high throughput setting avoiding radioactively labeled ligands.Fluorescence Confocal Microscopy in Live Cells.Having validated 28 and 29 as selective and useful fluorescent probes for CB 1 R pharmacology investigations, we next examined the potential for visualization of human CB 1 R on live SNAP-CB 1 R-HEK293TR cells by confocal microscopy (Figure 6).−85 For rigorous validation of selectivity and specificity, the experiments were performed side-by-side on tetracycline-inducible HEK293TR cells expressing CB 1 R and CB 2 R in comparison with parental HEK293TR cells without CBR expression.Probe 29 was able to selectively stain and visualize CB 1 R on the HEK cells (Figure 6A) within 10 min (see also Video S1).In addition to membrane CB 1 R, we observed staining of intracellular receptor pools of the CB 1 R positive HEK293TR cells (Figure 6A, white arrow, Figures S7 and  S8). 7,86Since 29 was shown to be an inverse agonist the possibility of ligand-induced internalization of membrane CB 1 R by 29 was excluded. 87Accordingly, probe 29 was able to passively permeate the outer cell membrane although exceeding typical drug-like parameters (see Table 4).This confirms the chameleonic behavior predicted by our MD simulation of probe 29.In contrast, no staining was observed on CB 2 R-HEK293TR or uninduced CB 1 R-HEK293TR (Figure 6B,C).Similarly, the uninduced CB 2 R-HEK293TR and HEK293TR cells without CBRs showed neither any staining nor unspecific background signal (Figures S3 and S4).The rapid staining (see Figures S5  and S6) and excellent CB 1 R-selectivity and specificity emphasize the real-time imaging capabilities of probe 29 and correlate with the selectivity determined in the radioligand binding assay.

■ CONCLUSIONS
In summary, by using drug-like CB 1 R ligands 5−10, we systematically explored the compatibility of our modular DEG-based CBR probe design approach.The screening from novel DEG ethyl esters 14−19 over PEG-linked compounds 22−27 to fluorescent probes 28−37 and 40−41 was guided by thorough pharmacological characterization and computational docking studies.
Our study showed that the DEG centerpiece can be used in combination with CB 1 R pharmacophoric units.Unfortunately, while CB 1 R binding and functional activity of the DEG ethyl esters was maintained, selectivity toward CB 1 R was strongly compromised for most structures.Upon the linker exploration, this trend was solidified except for diarylpyrazole series 23a−d, which turned into selective CB 1 R binders.Optimal linker length and attachment site were investigated by docking studies and confirmed by SAR studies using radioligand displacement assays.Among the tested fluorescent dyes, NBD was well tolerated without affecting the probes selectivity profile.In contrast, TAMRA installation had detrimental effects on the CBR binding affinities, except for 29 and 37. Most notably, throughout the exploration steps and among the tested structures, pyrrole-based compounds (14, 22a−c, and 28, 29) exhibited outstanding selectivity toward CB 1 R and tolerance for any modification.We characterized 28 and 29 as CB 1 R-selective inverse agonist fluorescent probes with applicability in TR-FRET kinetic and equilibrium CB 1 R ligand−receptor binding studies.In live cell confocal fluorescence microscopy, drugderived 29 showed rapid, highly selective, and specific staining of CB 1 R on HEK293TR cells.The observed membrane permeability of 29 was rationalized by in silico studies suggesting chameleonic effects.
Our novel building block strategy for probe design following reverse-design principles allowed for the fast accomplishment of well-validated, selective, and specific tools for fluorescencebased CB 1 R pharmacology.We believe that our CB 1 R probes will pave the way for a deeper and broader understanding of CB 1 R pharmacology in cannabinoid research.

■ EXPERIMENTAL SECTION
Radioligand Binding Assay.Cell Culture and Membrane Preparation.CHOK1hCB 1 _bgal and CHOK1hCB 2 _bgal cells (Dis-coverRx, Fremont, CA, USA) were cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham supplemented with 10% fetal calf serum, 1 mM glutamine, 50 μg/mL penicillin, 50 μg/mL streptomycin, 300 mg/mL hygromycin, and 800 μg/mL geneticin in a humidified atmosphere at 37 °C and 5% CO 2 .Cells were subcultured twice a week at a ratio of 1:20 on 10 cm plates by trypsinization.For membrane preparation, the cells were subcultured with a ratio of 1:10 and transferred to 15 cm ⌀plates.The cells were collected by scraping in 5 mL phosphate-buffered saline (PBS) and centrifuged at 1000g for 5 min.Pellets derived from 30 plates were combined and resuspended in 20 mL cold Tris−HCl, MgCl 2 buffer (50 mM Tris−HCl (pH 7.4), 5  mM MgCl 2 ).The cell suspension was homogenized using an UltraTurrax homogenizer (Heidolph Instruments Schwabach, Germany).Membranes and cytosolic fractions were separated by centrifugation in a Beckman Optima LE-80K ultracentrifuge (Beckman Coulter Inc., Fullerton, CA, USA) at 100,000g for 20 min at 4 °C.The supernatant was discarded.The pellet was resuspended in 10 mL cold Tris−HCl, MgCl 2 buffer, and homogenization and centrifugation steps were repeated.The membranes were resuspended in 10 mL cold Tris− HCl, MgCl 2 buffer.Aliquots of 100 μL were stored at −80 °C until further use.The protein concentration was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).
Data Analysis.All experimental data were analyzed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA).The data were normalized to % specific radioligand binding, where total binding is 100% and nonspecific binding is 0%.Nonlinear regression for one-site was used to determine the IC 50 values from the full curve [ 3 H]CP55940 displacement assays.The pK i values were obtained using the Cheng− Prusoff equation: 88 , where [L] is the exact concentration [ 3 H]CP55940 determined per experiment and the K D is the dissociation constant of [ 3 H]CP55940, which is 0.84 and 0.48 nM for CB 1 R and CB 2 R, respectively (data not shown).All data were obtained from at least three separate experiments performed in duplicate.
HTRF cAMP Assay for CB 1 R.The homogeneous time-resolved fluorescence (HTRF) cAMP assay was conducted following the manufacturer's protocol for the cAMP-G s Dynamic kit.Briefly, the CHO cell line stably overexpressing CB 1 R was cultured in Ham's F12 supplemented with 10% FBS, 10 μg/mL blasticidin, and 400 μg/mL zeocin.For the CB 1 R agonist or inverse agonist, dissociated cells were resuspended in Ham's F12 and dispensed into 384-well low-volume plates at 6000 cells/5 μL per well.The cells were then stimulated with compounds diluted in stimulation buffer (2.5 μL/well) for 15 min at room temperature, followed by the addition of 2.5 μL 5 μM forskolin or 2.5 μL 5 μM forskolin with 100 nM CP55,940 for antagonists.After 15 min, reactions were stopped by the 1× cAMP-d 2 conjugate in lysis buffer (5 μL/well), followed by 1× anti-cAMP cryptate conjugate in lysis buffer (5 μL/well).Following a 1 h incubation at room temperature, the plates were read in a Revvity Envision reader for time-resolved fluorescence resonance energy transfer detection at 620 and 665 nm.The HTRF ratio versus compound concentrations was plotted using Prism 8.1 (GraphPad).HTRF ratio = (signal 665 nm/ signal 620 nm) × 10 4 .All HTRF ratio data sets of test compounds were normalized to the E max of CP55,940 (100%) or AM281 (−100%) and obtained the means ± standard error of the mean (SEM) of three independent experiments performed in technical replicates.
TR-FRET Assay.Cell Culture.HEK293TR cells were maintained in a humidified environment at 37 °C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) containing blasticidin (5 μg/mL; Invitrogen) and (Zeocin; 20 μg/ mL; Invitrogen).For inducible expression, a SNAP-tagged human CB 1 R cDNA (in TR-FRET experiments, a truncated CB 1 R variant, CB 1 R 91-472 was used to facilitate the FRET, and named based on the residues remaining after truncation) in pcDNA4/TO was introduced through transfection, using PEI into HEK293TR cells (Invitrogen, which express Tet repressor protein to allow inducible expression).A mixed population stable line was selected by resistance to blasticidin (TR vector, 5 μg/mL) and Zeocin; (receptor plasmid, 20 μg/mL).For receptor-inducible expression, cells were seeded into T175 flasks, grown to 70% confluence, and DMEM containing 1 μg/mL tetracycline added.24 h later, cells were labeled with SNAP-Lumi4-Tb (CisBio) and membranes prepared as described in detail below.
Terbium Labeling of SNAP-Tagged CB 1 R HEK293-TR Cells.Cell culture medium was removed from the T175 flasks containing confluent adherent CB 1 R HEK293-TR cells.Cells were washed 1× in PBS (GIBCO Carlsbad, CA), followed by 1× Tag-lite labeling medium (LABMED, CisBio) to remove the excess cell culture media, and then 10 mL of LABMED containing 100 nM of SNAP-Lumi4-Tb was added to the flask and incubated for 1 h at 37 °C under 5% CO 2 .Cells were washed 1× in PBS (GIBCO Carlsbad, CA) to remove the excess of SNAP-Lumi4-Tb, then detached using 5 mL of GIBCO enzyme-free Hank's-based cell dissociation buffer (GIBCO, Carlsbad, CA), and collected in a vial containing 5 mL of DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum.Cells were pelleted by centrifugation (5 min at 350g), and the pellets were frozen to −80 °C.
Membrane Preparation.All steps were conducted at 4 °C to avoid tissue degradation.Cell pellets were thawed and resuspended using icecold buffer containing 10 mM HEPES and 10 mM EDTA, pH 7.4.The suspension was homogenized using an electrical homogenizer Ultra-Turrax (Ika-Werk GmbH & Co. KG, Staufen, Germany) and subsequently centrifuged at 1200g for 5 min.The pellet obtained then, containing cell nucleus and other heavy organelles, was discarded, and supernatant was centrifuged for 30 min at 48,000g at 4 °C (Beckman Avanti J-251 Ultracentrifuge; Beckman Coulter, Fullerton, CA).The supernatant was discarded, and the pellet was resuspended using the same buffer (10 mM HEPES and 10 mM EDTA, pH 7.4) and centrifuged for a second time for 30 min as described above.Finally, the supernatant was discarded, and the pellet resuspended using ice-cold 10 mM HEPES and 0.1 mM EDTA, pH 7.4.Protein concentration determination was carried out using the bicinchoninic acid assay kit (Sigma-Aldrich) and using BSA as a standard.The final membrane suspension was aliquoted and maintained at −80 °C until required for the binding assays.
Fluorescent Ligand-Binding Assays.All fluorescent ligand binding experiments were conducted in white 384-well Optiplate plates, in assay binding buffer, either Hanks Balanced Salt Solution (HBSS), 5 mM HEPES, 0.5% BSA, 0.02% pluronic F-127 pH 7.4, and 100 μM GppNHp.GppNHp was included to remove the G protein-coupled population of receptors that can result in two distinct populations of binding sites in membrane preparations since the Motulsky−Mahan model is only appropriate for ligands competing at a single site.In all cases, nonspecific binding was determined by the presence of 10 μM Rimonabant.
Determination of Fluorescent Ligand Binding Kinetics and Equilibrium Affinity.To accurately determine the association rate (k on ) and dissociation rate (k off ) values, the observed rate of association (k obs ) was calculated using at least six different concentrations of fluorescent ligand.The appropriate concentration of fluorescent ligand binding was incubated with human CB 1 R HEK293-TR cell membranes (0.5 μg per well) in assay binding buffer (final assay volume, 40 μL).The degree of fluorescent ligand bound to the receptor was assessed at multiple time points by HTRF detection to allow for the construction of association kinetic curves.The resulting data were globally fitted to the association kinetic model (eq 1, see Signal Detection and Data Analysis section below) to derive a single best-fit estimate for k on and k off as described under data analysis.Saturation analysis was performed at equilibrium, by simultaneously fitting total and nonspecific (NSB) binding data (eq 2, see Signal Detection and Data Analysis section below) allowing for the determination of fluorescent ligand binding affinity.
Competition Binding.To determine the affinity of CB 1 R-selective ligands, we used a simple competition kinetic binding assay.This approach involves the simultaneous addition of both fluorescent ligand and competitor to the CB 1 R preparation.Compounds were added simultaneously with increasing concentrations of the unlabeled compound to CB 1 R cell membranes (0.5 μg per well) in 40 μL of assay buffer in a 384-well Optiplate incubated at room temperature with orbital mixing.The degree of fluorescent ligand bound to the receptor was assessed at equilibrium by HTRF detection.Nonspecific binding was determined as the amount of HTRF signal detected in the presence of Rimonabant (10 μM) and was subtracted from total binding, to calculate specific binding for construction of IC 50 curves.
Signal Detection and Data Analysis.Signal detection was performed on a PHERAstar FSX (BMG Labtech, Offenburg, Germany).The terbium donor was always excited with four laser flashes at a wavelength of 337 nm.TR-FRET signals were collected at 590 (acceptor) and 620 nm (donor) when using the orange acceptor fluorescent ligand or at 520 (acceptor) and 620 nm (donor) when using the green acceptor fluorescent ligand.HTRF ratios were obtained by dividing the acceptor signal by the donor signal and multiplying this value by 10,000.All experiments were analyzed by nonregression using Prism 8.0 (GraphPad Software, San Diego, USA).Fluorescent ligand association data were fitted as follows to a global fitting model using GraphPad Prism 8.0 to simultaneously calculate k on and k off using the following equation where k obs equals the observed rate of ligand association and k on and k off are the association and dissociation-rate constants, respectively, of the fluorescent ligand.In this globally fitted model of tracer binding, tracer concentrations [L] are fixed, k on and k off are shared parameters, while k obs is allowed to vary.Here, Y is the level of receptor-bound tracer, Y max is the level of tracer binding at equilibrium, X is in units of time (e.g., min), and k obs is the rate in which equilibrium is approached (e.g., min −1 ).Saturation binding data were analyzed by nonlinear regression according to a one-site equation by globally fitting total and NSB.Individual estimates for the fluorescent ligand dissociation constant (K D ) were calculated using the following equations where L is the fluorescent ligand concentration Fitting the total and NSB data sets globally (simultaneously), sharing the value of slope, provides one best-fit value for both the K D and the B max .Competition displacement binding data were fitted to sigmoidal (variable slope) curves using a "four-parameter logistic equation" (log IC X) hill coefficient 50 (3) IC 50 values obtained from the inhibition curves were converted to K i values using the method of Cheng and Prusoff. 88 Imaging Experiments.Cell Culture.Human CB 1 R HEK293TR cells (same transfected cell line as for TR-FRET binding assay was used, see above) were maintained in a humidified environment at 37 °C and 5% CO 2 in DMEM with 10% FBS containing blasticidin (5 μg/mL; Invitrogen) and (Zeocin; 20 μg/mL; Invitrogen).
Methodology.Cells were plated onto a 384-well microplate (PhenoPlate, Revvity), at a density of 3500 cells/well (40 μL) with 1 μg/mL tetracycline for receptor-inducible expression and incubated for 48 h.The cell nuclei were stained using 0.9 μM Hoechst 33342 for 1 h incubation.After replacement of medium to serum-free conditions without Phenol red (20 μL), fluorescent probes were added (10 μL) and tested at 250 nM concentration.In case of blocking experiments, nuclei-stained cells were incubated with inhibitors (5 μM) for 30 min under serum-free conditions before probe administration.Confocal live cell imaging was performed using the Opera Phenix High Content Screening System (Revvity) at 22 °C.The probe fluorescence was monitored by kinetic measurements of 10 min with a break for probe administration.The fluorescence of one image per sample was captured using a water immersion objective (63×, NA 1.15, field of view 0.21 × 0.21 mm) at each time point.Probe detection was realized using the appropriate laser for excitation and filter for fluorescence emission.Image acquisition parameters, including laser power, offset, and gain settings, were kept constant.
Computational Docking.The previously reported X-ray diffraction structure for CB 1 R complexed with the CB 1 R antagonist, AM6538 (PDB: 5TGZ), 56 was used as a template to dock CB 1 R compounds.Docking experiments were performed interactively using MOE software (Chemical Computing Group) with default settings [Molecular Operating Environment (MOE), 2022.02;Chemical Computing Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2022].The most reasonable docking pose with respect to molecular interactions and internal conformational strain was energy-minimized within the binding pocket.Adjacent amino acid side chains were energy-minimized without restraints.The resulting docking poses were checked for consistency with the available structure−activity relationship (SAR) information.Visualization was performed using Maestro Schrodinger.
Photophysical Characterization.Absorbance/Emission Determination.50 μL of 10 μM solution of compound 28−37 and 40−41 in PBS (pH = 7.4) in the presence of 0.1% (v/v) DMSO was placed in a Corning 384-well Polystyrene microplates and the UV/vis absorbance spectra were first recorded in the wavelength range of 300−800 nm (scan step 5 nm) to determine the wavelength with the maximal absorbance signal, which was later used for excitation of the corresponding compound and fluorescent emission signal measurements.All measurements were performed at room temperature using a Tecan Safire II UV−vis fluorescence and absorbance plate reader.
Quantum Yield Determination.The absolute quantum yield was determined using a HAMAMATSU PHOTONICS K.K Absolute PL Quantum Yield Spectrometer with xenon lamp bulb L11562.For this purpose, 3 mL of 100 nM solution of compound in PBS (pH = 7.4) in the presence of 0.1% (v/v) DMSO were placed into a quartz cuvette with a rod (Size: 12.5 × 12.5 × 140 mm).After excitation, the quantum yield was recorded with the supplier's software version 4.6.0CD-ROM and reported as percentage.
Synthesis Procedures and Analytical Data for the Compounds.Reactions with air or moisture-sensitive substances were carried out under an inert atmosphere of nitrogen with the help of the Schlenk technique, if not otherwise indicated.All chemicals were purchased from commercial suppliers as received unless otherwise specified.(47) were synthesized according to the synthetic routes describes below.Compound names are derived from Chemdraw and are not necessarily identical to the IUPAC nomenclature.For thin layer chromatography aluminum backed silica gel plates were used (silica gel 60 F 254 from E. Merck), visualizing with UV light (λ = 254 nm).Microwave heating of reactions was carried out on a Biotage Initiator + apparatus.Chromatographic separations were carried out using Biotage Isolera One apparatus or Combiflash NextGen 300+ apparatus with RediSepRf columns from Teledyne Isco.High-performance liquid chromatography (HPLC) separations were carried out using a Gilson PLC 2050 system, a Gilson PLC 2250 or a Shimadzu system with the following components: CBM20A, LC20AP, SPD, 20A, and FC200Al.The Gilson systems were used with an automated gradient optimizer.As the stationary phase, a Macherey-Nagel VP 250/21 Nucleodur 100-7 C18Ec column or a Macherey-Nagel VP 250/10 Nucleodur 100-5 C18Ec column was used.As the mobile phase, ACN/water with 0.1% TFA as an acidic modifier or ACN/water was used.The analytical data was obtained with the help of the following equipment: 1 H and 13 C NMR spectra were recorded at either Bruker AV 300 (295 K, 300 MHz, 75 MHz), Bruker AV 600 (300 K, 600 MHz, 151 MHz), or Bruker AV 750cryo (300 K, 750 MHz, 189 MHz) spectrometers in CDCl 3 , MeOD, ACN-d 3 or DMSO-d 6 as solvents.Spin multiplicities were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) doublet of doublet (dd), doublet of triplet (dt), doublet of quartet (dq), and broad-singlet (br s).Coupling constants ( n J, whereby n equals the number of bonds between the coupled nuclei) were recorded in Hz.All 13 C NMR-spectra were recorded with 1 H-broad-band decoupling.All chemical shifts are reported as found in ppm (δ) relative to tetramethylsilane (δ = 0.00 ppm) and were calibrated with respect to their deuterated solvents. 89NMR data were analyzed with MNova.Analytical HPLC-MS and purity analyses were performed with Agilent 1260 series HPLC system employing a DAD detector (at 300, 254, and 220 nm) equipped with Agilent Technologies 6120 Quadrupole LC/ MS in electrospray positive and negative ionization modes (ESI-MS).A Thermo Accuore RP-MS (30 × 2.1 mm, 2.6 μm) column was used with a flow rate 0.8 mL/min in combination with the following separation conditions: 0.1% formic acid in water (solvent A); 0.1% formic acid in ACN (solvent B); system (1) 5% B for 0.5 min, from 5 to 95% B in 6.5 min, 95% B for 1 min (stop point at 8 min); system (2) 5% B for 0.2 min, from 5 to 95% B in 0.9 min, 95% B for 1.4 min (stop point at 2.5 min).Data analysis was performed with ChemStation software.All compounds are >95% pure by HPLC.High-resolution mass spectrometry (HRMS) analyses were carried out on Agilent Technologies 6530 Accurate Mass Q-ToF LC/MS linked to Agilent Technologies HPLC 1260 Infinity II and HRMS results are reported in m/z.

Scheme 1 .
Scheme 1.General Synthetic Routes for the Construction of Evaluated Ligands a

Figure 3 .
Figure 3. Docking poses of representative pyrrole-based CB 1 R ligand 14 and DEG probe precursor 22b in the CB 1 R inactive state (light gray, docked in PDB ID: 5TGZ, X-ray diffraction, 2.80 Å). 56 (A) Docking pose of novel DEG ethyl ester ligand 14 (bright yellow) located in the binding pocket of CB 1 R with the ethyl ester group pointing toward the N-terminal site.(B) Linker installation on 14 via the carboxy-terminal amide bond of DEG is a reasonable strategy based on the docking structures.(C) DEG probe precursor 22b with n = 2 (orange).(D) Linker reaches the CB 1 R extracellular site through trans-membrane helices (TM) 1 and 2. For docking poses of 15−19, 23b, and 24−27 and a detailed description of the docking studies, see Supporting Information.

aK
i (nM) values obtained from [ 3 H]CP55,940 displacement assays on CHO membranes stably expressing human CB 1 R or human CB 2 R. Values are means of three independent experiments performed in duplicate.b The activity levels (EC 50 or IC 50 ) of 14−19 were measured using cells stably expressing hCB 1 R in homogeneous time-resolved fluorescence (HTRF) cAMP assay.The data are the means of three independent experiments performed in technical replicates.c Maximum effect (E max in %) was normalized to reference full agonist CP55,940.n.a.denotes no activity.All data with standard error of mean are given in the Supporting Information.

Figure 4 .
Figure 4. Conformational analysis of 29.(A) Equilibrium of 5/6-TAMRA isomers as spirolactone (closed) and zwitterionic (open) form.(B) Violin plot of the 3D PSA distribution of the four possible isomers (open/closed, 5/6-isomer) of 29 in water and octane obtained by MD simulation (50 ns).Drug-like PSA cutoff 140 Å 2 and tPSA of spirolactone and zwitterion form are indicated as dotted lines.Mean 3D PSA of each isomer is indicated as black line in the violin plot.(C) Intramolecular hydrogen bond formation observed in MD simulation (50 ns) of the four possible isomers (open/ closed, 5/6-isomer) of 29 in water and octane.Mean hydrogen bond interactions represented as bar chart ± SEM.

Figure 5 .
Figure 5. TR-FRET binding assays using HEK293TR-hCB 1 R cell membranes.(A) Observed association curves of TAMRA probe 29 to hCB 1 R. (B) Saturation binding analysis of 29 to hCB 1 R after 60 min.(C) Competition binding using 29 (300 nM) as a tracer with increasing concentrations of CB 1 R ligand 6 and HU210.(D) Competition binding using NBD probe 28 (60 nM) as a tracer with increasing concentrations of CB 1 R ligand 6 and HU210.Kinetic and equilibrium data were fitted to the equations described in the Supporting Information to calculate K D , k on , and k off values for fluorescent and unlabeled ligands.Data are presented as mean ± SEM, N = 3.

Table 1 .
Binding Affinities and Functional Activity of CB 1 R DEG Ligands

Table 2 .
Binding Affinities and Functional Activity of the N-Boc-Protected DEG Probe Precursors a K i (nM) values obtained from [ 3

Table 4 .
Calculated Physicochemical Descriptors of DEG Ethyl Ester Ligand 14 and Probe 29 Isomers as Spirolactone and Zwitterion Forms by Chemoinformatic Tools

Table 5 .
HTRF Binding Parameters of CB 1 R Probe 29 and Unlabeled Ligands a compd.k on (10 6 M −1 min −1 ) k off (min −1 ) RT (min) kinetic K D (nM) K i (nM) b K i (nM) c a Data are presented as mean, N = 3. b Probe 29 (300 nM) used as a tracer.c Probe 28 (60 nM) used as a tracer.RT: residence time.All data with standard error of mean are given in the Supporting Information.